The long term goal is to develop a vaccine that prevents toxoplasmosis, including transmission from mother to child and reduction or elimination of organisms during initial acute infection. Work toward this goal will be accomplished using a rational approach based on understanding and using only epitopes that induce a protective immune response, without extraneous epitopes that may be harmful, and optimizing adjuvants. Specific aims are: 1. Identify optimal adjuvants and delivery, characterizing and optimizing peroral bilosome delivery systems for DMA vaccines; 2.Characterize protective effect, and when there is protection, immune responses produced by SAG1 or ROP2 or GRA2 or 3 or 6 or 7 or a bradyzoite antigen (BLKAg), and for oocyst challenge a sporozoite antigen (SpAg) vaccination. HLA A2.1, HLA A3/11 and HLA B7 mice, will be vaccinated with DMA constructs containing parasite antigens, with and without adjuvant(s) or bilosomes or MVA for boost; 3. Create multiepitope vaccines based on epitopes identified from those proteins which confer protection; 4. In later studies, test the most promising constructs in (a) peroral bradyzoite or oocyst challenges, (b) challenges with hypervirulent, recombinant and a Brazilian strain of T. gondii, and (c) in congenital and ocular models. Studies initially will define the best adjuvant(s) of 6 cytokine eliciting or cytokine encoding constructs or delivery with bilosomes or MVA boost construct using T. gondii SAG1. These will be used to vaccinate HLA A2 transgenic mice with DNA from clonal type 1 or II parasites and challenge with the homologous clonal type of parasite. Adjuvants that will be tested include DNA constructs which encode each of the following separately: IL -1Beta,-2,-12,-15,and -18; delivery within an attenuated vaccinia construct following an injection within the DNA construct; and delivery in bilosomes. Then, vaccines will be tested with DNA encoding T. gondii proteins that are candidates for protection with the best adjuvant(s) or means of delivery in HLA supermotif transgenic B7, and A*201 and A3/11 mice (HLA supermotifs present in ~90% of humans). Proteins were selected based on data available about peptides that enter MHC Class I pathways and confer protection in inbred strains of mice, including SAG1, ROP2, GRA2, GRA3, GRA6, GRA7, BLKAG, and SpAg. When protective proteins in each HLA supertype mouse are identified, epitope-encoding constructs will be designed following the principles of vaccine optimization defined by the Sette/Alexander group. This work will provide a foundation to develop a vaccine protective against toxoplasmosis for humans as well as a paradigm for vaccines to protect against other pathogens. [unreadable] [unreadable] [unreadable]